Methods for diagnosing and treating eye-length related disorders

ABSTRACT

The invention provides to methods for diagnosing eye-length related disorders, including myopia. The invention also provides methods for treating and limiting eye-length related disorders, including myopia. In addition, the invention provides certain haplotypes associated with eye-length related disorders, including myopia and Bornholm Eye Disease.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.14/581,116 filed Dec. 23, 2014, which is a continuation of U.S.application Ser. No. 13/349,877 filed Jan. 13, 2012, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/432,984filed Jan. 14, 2011, incorporated by reference herein in its entirety.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This work was supported by the National Institutes of Health grantsR01EY09620, P30 FY01931, and P30EY01730. The U.S. government has certainrights in the invention.

FIELD OF THE INVENTION

The invention relates to methods for detecting and treating eye-lengthrelated disorders, including myopia. In addition, the invention relatesto certain haplotypes associated with eye-length related disorders.

BACKGROUND

In a process termed emmetropization, the growth of eye length isregulated by visual experience to match the eye's optics and tocompensate for genetic variation in corneal/lens curvature and power.High acuity photopic vision and, thus, the signals that guideemmetropization are initiated by light absorption photopigments found inthe long wavelength (L) and middle-wavelength (M) sensitive conephotoreceptors. Changes in the pattern of light and dark in the retinalimage that characterize blurred versus sharply focused images aremonitored by a biological process to stop eye growth when the correctlength for coordinated plano (neutral) optics is reached. However, inmyopic individuals, the relative axial length of the eye to overall eyesize continues to increase during development, past a length thatprovides near-optimal focusing of distant objects, leading toincreasingly pronounced myopia.

The rate of incidence of myopia is increasing at alarming rates in manyregions of the world. Until recently excessive reading during childhoodwas believed to be the only identifiable environmental or behavioralfactor linked to the occurrence of myopia, although genetic factors weresuspected. Limiting reading (and encouraging more outdoor activity) arepresently the only practical techniques for preventing excessive eyelengthening in children, and corrective lenses, including glasses andcontact lenses, represent the primary means for ameliorating eye-lengthrelated disorders, including myopia. While these measures opticallycorrect the refractive errors associated with eye-length relateddisorders they do not address the underlying cause which is excessivegrowth of eye length.

Thus, there remains a need for methods of detecting a susceptibility toan eye-length related disorder, and treatments for such individuals thatwould prevent excessive eye lengthening.

SUMMARY OF THE INVENTION

The invention provides a method for determining the myopic potential ofa patient comprising: testing a biological sample obtained from thepatient to determine the L:M opsin gene haplotype of the patient; andcorrelating the haplotype with a predicted spherical equivalentrefraction. In another aspect, the method further comprises the stepsof: determining the L:M cone ratio in an eye of the patient; andcorrelating the L:M opsin gene haplotype and the L:M cone ratio with apredicted spherical equivalent refraction.

The invention also provides a method for diagnosing susceptibility of apatient to an eye-length related disorder, the method comprising:testing a biological sample obtained from the patient to determine theL:M opsin gene haplotype of the patient; and correlating the haplotypewith a predicted spherical equivalent refraction; wherein the patient issusceptible to an eye-length related disorder if the predicted sphericalequivalent refractive error (measured in diopters) has a negative power.In one aspect, the method further comprises the steps of: determiningthe L:M cone ratio in an eye of the patient; and correlating the L:Mopsin gene haplotype and the L:M cone ratio with a predicted sphericalequivalent refraction.

The invention further provides a method for diagnosing susceptibility ofa patient to an eye-length related disorder, the method comprisingtesting a biological sample obtained from a patient for a particularcombination of amino acids encoded by the patient's L opsin gene or Mopsin gene, wherein the patient is susceptible to an eye-length relateddisorder if one of the amino acid combinations shown in Table 1 ispresent.

In addition, the invention provides a method of treating an eye-lengthrelated disorder comprising: testing a biological sample obtained fromthe patient to determine the L:M opsin gene haplotype of the patient;determining the L:M cone ratio in an eye of the patient; correlating thehaplotype and the L:M cone ratio with a predicted spherical equivalentrefraction; providing the patient with a therapeutic device comprising awavelength-dependent filter if the patient's predicted sphericalequivalent refractive error has a negative, power.

In one aspect, the wavelengths filtered by the wavelength-dependentfilter are selected based on the L:M opsin gene haplotype and the L:Mcone ratio of the patient.

In another aspect, a therapeutic device used in a method of theinvention is a pair of spectacles comprising blur-inducing lenses. Incertain aspects, the blur-inducing lenses induce blurring by one or moreof: small bumps or depressions in one or both surfaces of the lenses;inclusions within the lenses of a material different from the lensmaterial; incorporation of higher-level aberrations in the lenses;providing an increased correlation between the activities of neighboringcone photoreceptors by one or both lenses; and coatings or films appliedto one or both surfaces of the lenses to produce diffusive ordiffractive blur.

In yet another aspect, a therapeutic device used in a method of theinvention comprises blur-inducing, contact lenses. In certain aspects,the blur-inducing contact lenses induce bluffing by one or more of:inclusions within the lenses of a material different from the lensmaterial; incorporation of higher-level aberrations in the lenses; andcoatings or films applied to one or both surfaces of the lenses thatproduce blur by diffusion, diffraction or light scattering.

In certain aspects, the L:M opsin gene haplotype identified in a methodof the invention is one of haplotypes 1 to 13 as set forth in Table 1.

The invention also provides a microarray for determining susceptibilityof a patient to an eye-length related disorder comprising a set ofallele specific oligonucleotides capable of identifying at least onehaplotypes 1 to 13 as set forth in Table 1.

The invention further provides kits for determining whether a patient issusceptible to an eye-length related disorder. In one aspect, a kit ofthe invention comprises: at least one pair of oligonucleotides that canidentify at least one of haplotypes 1 to 13 as set forth in Table 1; andinstructions for use. In another aspect, a kit of the inventioncomprises an assay for detecting at least one of haplotypes 1 to 13 asset forth in Table 1.

In another aspect, the present invention provides methods for limitingintroduction of refractive error in a subject's eye caused by exposureto display screens, comprising the subject wearing a therapeutic opticaldevice that comprises a wavelength-dependent filter capable ofpreferentially blocking red light emanating from the display screenprior to entry into the subject's eye, thereby limiting introduction ofrefractive error in the subject's eye.

In a further aspect, the present invention provides methods for limitingdevelopment of an eye-length related disorder in a subject, comprisingthe subject wearing a therapeutic optical device that comprises awavelength-dependent filter capable of preferentially blocking red lightemanating from a display screen prior to entry into the subject's eye,thereby limiting development of an eye-length related disorder in thesubject. In one embodiment, the eye-length related disorder comprisesmyopia.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Averaged, adaptive optics retinal images of the cone mosaic ofparticipants with LIAVA variants (B, C, D) compared with a normalcontrol (A). For subjects shown in B, C, & D cones expressing the LIAVAvariant had a low reflectance compared to normal cones and appear asdark area in the mosaic. There was large variability in the proportionof cones expressing the LIAVA variant. B, C & D have low, medium andhigh proportions of cones expressing the myopia-genic variant whichcorrelates with axial length (E) and also with refractive error.

FIG. 2. Association between axial length and cone ratio for differentethnic groups. There was a high positive correlation between L:M coneratio and axial length (and incidence of myopia) across ethnic groups.

FIG. 3. (A) Myopic potential of 13 different UM photopigment haplotypes,from 159 males, arranged in order of increasing myopic potential. Thenumber of individuals with each haplotype is given at the right.Haplotype designations use the single letter amino acid code:M=methionine, 1=isoleucine, S=serine, V=valine, A=alanine, andL=leucine. Average SER is the mean spherical equivalent refractioncalculated for the most myopic half of the subjects for each haplotype,±1 SEM. (B) Predicted versus observed spherical equivalent refraction(SER) for 11 subjects with haplotypes corresponding to those describedin (A). The L:M cone ratio was estimated for each subject and isexpressed as the percentage of L plus M cones that are L.

FIG. 4. (A) Myopic shift produced by exposure to the red light for 2hours per day. Axial lengths were measured for each subject before theonset of the experimental procedure. Subsequently, each subject played ablack and white video game for 2 hours per day while wearing goggleswith the right lens untinted and the left lens tinted so that the Lcones are activated much more than M cones. (B) Normalized axial lengthmeasurements as a function of time for 20 eyes wearing the experimentallens and (C) for 20 fellow eyes that served as the controls for eachexperimental eye. Black lines with error bars represent the averages forall eyes (error bars±2 SEM). The experimental lenses significantlyreduced the rate of eye growth of myopic children. (D) Growth rate ofeyes wearing the experimental lens are to the left, and for eyes wearingthe control lens are to the right.

DETAILED DESCRIPTION OF THE INVENTION

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

The following definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition or a dictionary known to those of skill inthe art, such as the Oxford Dictionary of Biochemistry and MolecularBiology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”. Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

In certain embodiments, the invention provides methods that can be usedto determine the benefit of a preventative treatment for an eye-lengthdisorder and to determine the appropriate prescription ofcharacteristics of preventative optics for a patient who is identifiedas having a susceptibility to an eye-length related disorder. Asdiscussed herein, such preventative optics include spectralcharacteristics and/or dispersive properties that can prevent eye-lengthgrowth, which if left uncontrolled would lead to an eye-length relateddisorder.

In one embodiment, the invention provides a method for diagnosingsusceptibility of a patient to an eye-length related disorder, themethod comprising: testing a biological sample obtained from the patientto determine the patient's L:M opsin gene haplotype, and correlating thehaplotype with a predicted spherical equivalent refraction; wherein thepatient is susceptible to an eye-length related disorder if thepredicted spherical equivalent refraction is a negative diopter.

As used herein, the term “correlating” refers to the step of using thecombination of information about a patient's cone ratio and opsinhaplotype in order to determine the susceptibility of the patient to aneye-length related disorder as shown and discussed herein.

As used herein, the phrase “eye-length related disorder” includes, butis not limited to, myopia.

In one embodiment, the L:M opsin gene haplotype is determined byidentifying the nucleotide sequence of a patient's DNA to determine thepatient's Xq28 opsin gene locus haplotype. The haploytpe can bedetermined by identifying, the nucleotide sequence of exons 2, 3, and 4of the OPN1LW and OPN1MW genes. As discussed herein, the haplotypes arecreated by the amino acids encoded by codons 65, 111, 116, 153, 171,178, 180, 230, 233, and 236 of the OPN1LW and OPN1MW genes. In aparticular embodiment, the haplotype is determined by the amino acidsencoded by codons 153, 171, 178, and 180 in exon 3 and codon 236 in exon4. In a preferred embodiment, the L:M opsin gene haplotype is one of the13 haplotypes shown in Table 1, which are shown herein for the firsttime as being associated with myopia (see Examples and FIG. 3A). Thus,if a patient has one of the 13 haplotypes identified in Table 1, thatpatient is diagnosed as being susceptible to an eye-length relateddisorder. In particular, a patient having one of the haplotypes shown inTable 1 is diagnosed as being susceptible to myopia. In one embodiment,a patient is diagnosed as being susceptible to myopia if one of thevariant amino acid combinations shown in Table 1 associated with theL-opsin gene is identified in the patient. In another embodiment, apatient is diagnosed as being susceptible to myopia if one of thevariant amino acid combinations shown in Table 1 associated with theM-opsin gene is identified in the patient.

TABLE 1 Myopia Haplotypes L-OPSIN M-OPSIN Codons Codons 153 171 178 180236 153 171 178 180 1 M I I S M M V V A 2 M V I S M M V V A 3 L V I S MM V V A 4 M V I S M M V I A 5 L V I A M M V I A 6 M V I A M M V I A 7 LV I S M L/M V I A 8 L V I S M M V I A 9 L I I S M M V V A 10 M V V A V MV I A 11 M V I S V M V V A 12 L V I S M L V I S 13 L V I A M L/M V I A

In another embodiment, the invention provides a method for diagnosingsusceptibility of a patient to an eye-length related disorder, themethod comprising: testing a biological sample obtained from the patientto determine the patient's L:M opsin gene haplotype, determining the L:Mcone ratio in an eye of the patient, and correlating the L:M opsin genehaplotype and the L:M cone ratio with a predicted spherical equivalentrefraction, wherein the patient is susceptible to an eye-length relateddisorder if the predicted spherical equivalent refractive error is anegative power (in diopters).

The L:M cone ratio can be determined using methods known to those ofskill in the art. For example, adaptive optics retinal imaging can beused as described herein, or an electroretinogram (ERG) (such as aflicker photometric ERG) and individualized cone spectra can be used.The L:M cone ratio measurement can also involve genetics, as describedfor example in Neitz and Neitz, J. Vis. 2:531-42, 2002. Anothernon-limiting example of measuring L:M cone ratio includes wide-fieldcolor multifocal ERG as described in Kuchenbecker et al., Vis. Neurosci.25(3):301-6, 2008. Another non-limiting example of measuring L:M coneratio includes measuring the ratio of red-to-green light perceived tohave the minimum flicker using psychophysical heterochromatic flickerphotometry as described in Gunther and Dobkins Vision Research42:1367-1378, 2002.

In one embodiment, the invention provides a method for determining themyopic potential of a patient comprising testing a biological sampleobtained from the patient to determine the patient's L:M opsin genehaplotype.

As used herein, the term “myopic potential” refers to the predictedspherical equivalent refraction associated with an L:M opsin haplotype,which correlates with the predicted degree of myopia that the patienthas or is likely to have. In particular, the myopic potential refers toa certain spherical equivalent refraction predicted based on thepatient's particular L:M opsin gene haplotype, as shown, for example, inFIG. 3A.

In a particular embodiment, myopic potential can be more specificallydetermined by measuring the patient's L:M cone ratio, and correlatingthe ratio with the spherical equivalent refraction predicted for theparticular L:M opsin gene haplotype. For example, as discussed in theexamples below, the L:M cone ratio can be determined for a patient thathas a certain L:M opsin haplotype, such as a haplotype shown in Table 1.The L:M cone ratio is determined, and a calculation is made to arrive atthe more specific predicted myopic potential. For instance, if a personhad haplotype 8 (FIG. 3A), their myopic potential is −4.5 diopters. Ifthat person had a 1:1 cone ratio they would be expected to have the full−4.5 diopters of refractive error. However, if he had nearly 100 percentL cones he would be expected to be nearly emmetropic. 75% L cones fallsmidway between a 1:1 cone ratio (50% L) and 100% L so a person withhaplotype 8 and 75% L cones would be predicted to have 50% of the SER(or −4.5/2=−2.25 diopters).

As used herein, the phrase “susceptibility to an eye-length relateddisorder” refers to the high likelihood of developing an eye-lengthrelated disorder, such as myopia, when a certain L:M opsin genehaplotype is present. In one embodiment, a patient is consideredsusceptible to an eye-length related disorder if one of the haplotypesshown in Table 1 is present, which are listed in order of increasingmyopic potential.

After identifying a patient that is susceptible to an eye-length relateddisorder and/or has a myopic potential associated with a negativediopter as described herein, an eye care provider can prescribe atreatment protocol and/or suggest certain behaviors intended to treat orreduce the myopic potential of the patient. For example, a patient maybe treated with a therapeutic device (as described herein, for example)or be given pharmacological intervention. In addition or instead of suchtreatments, a patient may be told to limit exposure to red light orgreen light (depending on the patient's particular variants) limitreading at a young age and spending more time doing activities outdoors.

The term “biological sample” as used herein includes, but is not limitedto, blood, saliva, cells from buccal swabbing, biopsies of skin,amniotic fluid, various other tissues and the like. Methods forpurifying or partially purifying nucleic acids from a biological samplefor use in diagnostic assays are well known in the art. The nucleic,acid can be, for example, genomic DNA, RNA, or cDNA. Genomic DNA can beisolated, for example, from peripheral blood leukocytes using QIAamp DNABlood Maxi Kits (Qiagen, Valencia, Calif.).

In another embodiment, the invention provides a method for diagnosingBornholm Eye Disease (BED) in a patient, the method comprising obtaininga biological sample from the patient and identifying the nucleotidesequence of the patient's L and M opsin genes, wherein the patient isdiagnosed as having BED if the patient has a normal opsin gene and avariant opsin gene. In a preferred embodiment, the variant opsin genecomprises Leucine at amino acid position 153 (L153), Valine at position171 (V171), Alanine at 174 (A174), Valine at 178 (V178), and Alanine at180 (A180) (“LVAVA”) or Leucine at amino acid position 153 (L153),Isoleucine at position 171 (I171), Alanine at 174 (A174), Valine at 178(V178), and Alanine at 180 (A180) (“LIAVA”) in either the L or M opsingene. In another embodiment, the second gene has the combination ofMethionine, Valine, Valine, Valine, and Alanine at amino acids atpositions 153, 171, 174, 178, and 180 (“MVVVA”).

The diagnostic methods of the invention involve the use of standardmolecular biology methods, including in one non-limiting embodiment thepolymerase chain reaction (PCR), to determine the L:M opsin genehaplotype of a patient. There are currently a variety of molecularbiological methods available that allow examination of the DNA sequencesof the L and M opsin genes. For example, gene fragments may be amplifiedusing the polymerase chain reaction (PCR). The genes can be separatelyand selectively amplified as described previously (Neitz et al., VisionResearch 35: 2395-2407, 1995).

Amplified gene fragments will preferably be subjected to one or more ofthe following procedures that provide information about the DNAsequence:

1) Direct DNA sequence of the PCR products as described previously (J.Neitz, M. Neitz and Grishok, supra, 1995).

2) Restriction digestion analysis (described previously in J. Neitz, M.Neitz and Grishok, supra, 1995).

3) Single strand conformation polymorphism or other similar procedures.The amplified DNA fragment is fluorescently or radioactively endlabeled, denatured into single strands, and the strands are separatedelectrophoretically. Based on the mobility of the strands in theelectric field, information about the DNA sequence can be deduced.

In another embodiment, the invention provides a method of treating aneye-length related disorder comprising: testing a biological sampleobtained from a patient to determine the L:M opsin gene haplotype of thepatient; determining the L:M cone ratio in an eye of the patient;correlating the haplotype and the L:M cone ratio with a predictedspherical equivalent refraction; providing the patient with atherapeutic device comprising a wavelength-dependent filter if thepatient's predicted spherical equivalent refraction is a negativediopter. In one embodiment, the opsin gene haplotype is one ofhaplotypes 1 to 13 as set forth in Table 1.

As discussed in International Patent Application Publication No. WO2010/075319, the entire contents of which are hereby incorporated byreference in their entirety, genetic variation in opsin genes affectsthe absorbance characteristics of the opsin photoreceptor protein. Thus,the wavelength-dependent filter utilized in a method of the invention isintended to filter light prior to entry into the eye in order to adjustthe effective absorbance spectrum of variant opsin photoreceptorproteins. In patients having a defective M photoreceptor protein, causedby a variant M-opsin gene, that absorbs less light than the normal Mphotoreceptor protein, the wavelength-dependent filter maypreferentially block red light. On the other hand, in patients having adefective L photoreceptor protein, caused by a variant M-opsin gene,that absorbs less light than the normal L photoreceptor protein, thewavelength-dependent filter may preferentially block green light.

In certain embodiments, the particular wavelength-dependent filterutilized in a method of the invention can be selected based on thepatient's L:M opsin gene haplotype, which identifies specificphotoreceptor variants and/or the patient's L:M cone ratio, whichidentifies the number of L photoreceptors relative M photoreceptorspresent in the patient's eye. Based on the particular L:M opsin genehaplotype and/or the L:M ratio, a filter can be designed to block and/ortransmit very specific wavelengths to restore relative absorptioncharacteristics of the defective photoreceptor proteins. Thus, theinvention further provides methods for customizing a therapeutic devicefor a particular patient based on the L:M opsin gene haplotype and/orthe L:M cone ratio of the patient. For example, if the patient had opsinvariants associated with more active red (M) cones, the filter could bedesigned to block red light; whereas if the patient had opsin variantsassociated with more active green (L) cones, the filter could bedesigned to block green light.

In certain embodiments, the therapeutic device comprises blur-inducinglenses, for example as described in International Patent ApplicationPublication No. WO 2010/075319. In one embodiment, the device is a pairof spectacles comprising blur-inducing lenses, where the blur isdesigned to reduce the relative activities between neighboring conephotoreceptors in the retina which has been shown herein to result insignals that stimulate the eye to grow in length abnormally. Theblur-inducing lenses can be made to induce blurring, for example, by oneor more of: small bumps or depressions in one or both surfaces of thelenses; inclusions within the lenses of a material different from thelens material; incorporation of higher-level aberrations in the lenses;and coatings or films that induce blur by light scatter, diffusion ordiffraction applied to one or both surfaces of the lenses.

In yet another embodiment, the therapeutic device comprisesblur-inducing contact lenses. The blur-inducing, contact lenses can bemade to induce blurring, for example, by one or more of inclusionswithin the lenses of a material different from the lens material;incorporation of higher-level aberrations in the lenses; providingprogressive negative corrections in one or both lenses from the centerof the lens to the bottom of the lenses; and coatings or films thatinduce blur by light scatter, diffusion or diffraction applied to one orboth surfaces of the lenses.

In one further aspect, the present invention provides methods forlimiting introduction of refractive error in a subject's eye caused byexposure to display screens, comprising the subject wearing atherapeutic, optical device that comprises a wavelength-dependent filtercapable of preferentially blocking red light emanating from the displayscreen prior to entry into the subject's eye, thereby limitingintroduction of refractive error, in the subject's eye.

In a still further aspect, the present invention, provides methods forlimiting development of an eye-length related disorder in a subject,comprising the subject wearing a therapeutic optical device thatcomprises a wavelength-dependent filter capable of preferentiallyblocking red light emanating from a display screen prior to entry intothe subject's eye, thereby limiting development of an eye-length relateddisorder in the subject. In one embodiment, the eye-length relateddisorder comprises myopia.

These methods can be used to limit damage to the eye caused by excessiveexposure to red-light from a screen display. In various non-limitingembodiments, the screen display may be a computer monitor, a tabletmonitor, a television screen, a handheld device screen, a video gamescreen, a head-mounted display screen, and a movie theater screen.

As used herein, “limiting” means one or more of (a) reducing theincidence of introduction of refractive error in a subject's eye and/orreducing the incidence of eye-length related disorders developing intreated subjects: (b) reducing the severity of subsequently developedrefractive error in a subject's eye and/or reducing the severity of asubsequently developed eye-length related disorder in the subject;and/or (c) limiting or preventing development of symptoms characteristicof refractive error in a subject's eye and/or an eye-length relateddisorder.

In each of these further aspects, the therapeutic optical device mayfurther comprise a blur-inducing lens, including but not limited tothose disclosed, in WO 2010/075319 and as disclosed above. In oneembodiment, the blur-inducing lens comprises a holographic diffuserapplied to the lens surface, for example, as described in the examplesbelow. The holographic diffuser can be used, for example, to spread theincident light rays from the display over a desired angle to produce aslight blur and thus reduce activity differences between adjacent cones.In any of these embodiments, the therapeutic optical device may be ofany suitable type, including but not limited to glasses/spectacles andcontact lenses.

Any suitable subject may be treated in these aspects, including children21 years of age or Younger, preferably between the ages of 3-21, 3-20,3-19, or 3-18. In another embodiment that can be combined with any ofthe above embodiments, wherein the subject is susceptible to aneye-length related disorder, such as myopia. This embodiment maycomprise treating any subject at risk as discussed in any of thepreceding disclosure. In one particular embodiment, the subject issusceptible to an eye-length related disorder if the subject has an L:Mopsin gene haplotype as set forth in Table 1.

In one embodiment, the invention provides kits that can be used, forexample, for eye-length related disorder diagnosis. In certainembodiments, a kit of the invention comprises a set of haplotypespecific oligonucleotides to identify the presence or absence of L:Mopsin gene haplotypes, such as those identified in Table 1. For example,a kit comprises: a set of primer pairs for amplifying portions of exons3 and 4 associated with the haplotypes described herein, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the haplotypes listed in Table1; a set of probes that can hybridize to portions of exons 3 and 4associated with the haplotypes described herein; and/or a microarray,such as a SNP chip. Primers and probes can be readily and easilydesigned by those skilled in the art by reference to a sequenceassociated with the portions of exons 3 and 4 associated with thehaplotypes described herein. Microarrays can also be easily and readilydesigned with oligonucleotides of the invention that correspond to theportions of exons 3 and 4 associated with the haplotypes describedherein. Alternatively, analysis could be done using a mass spectrometryinstrument (for example, a MassArray™ instrument) that allows genotypingat known polymorphic sites using specially designed PCR primers followedby mass spectrometry. This technique is suited to diagnosis ofconditions such as axial length disorders described here whose geneticunderpinnings are well understood. A MassArray™ primer extension processdetects sequence differences at the single nucleotide level. An initialround of PCR amplifies from genomic DNA a short length of DNAsurrounding the SNP. This is followed by single-base extensions of aprimer that anneals directly adjacent to the SNP. The primer is extendeddependent upon the template sequence, resulting in an allele-specificdifference in mass between extension products. This mass differenceallows differentiation between SNP alleles using MALDI TOF massspectrometry.

In another embodiment, the invention provides a mouse model of aneye-length related disorder as described in the Examples herein, whichComprises a variant green (L) photopigment protein associated withmyopia. The invention further provides a mouse model that expressesvariant red (M) and normal or variant green (L) photopigment proteins,wherein a variant protein has an amino acid sequence associated withmyopia. Such mice can be generated as described, for example, in theMethods provided herein. Such mice have been generated using the methoddescribed herein, wherein the heterozygous mice of the method comprisethe red and green photopigment proteins. In certain embodiments, a mousemodel of the invention can be used to test eye-length related disorderintervention, such as pharmacological or genetic intervention.

In certain embodiments, the present invention provides a machinereadable storage medium, comprising a set of instructions for causing adiagnostic device to measure a patient's L:M cone ratio or L:M opsingene haplotype. In other embodiments, the invention provides a machinereadable storage medium that comprises instructions for causing aprocessor to execute automated method steps for correlating a patient'sL:M opsin gene haplotype and L:M cone ratio to determine an appropriateprescription of characteristics of preventative optics for a patient whois identified as having a susceptibility to an eye-length relateddisorder. As used herein the term “computer readable storage medium”includes magnetic disks, optical disks, organic memory, and any othervolatile (e.g., Random Access Memory (“RAM”)) or non-volatile (e.g.,Read-Only Memory (“ROM”)) mass storage system readable by the CPU. Thecomputer readable medium includes cooperating or interconnected computerreadable medium, which exist exclusively on the processing system or bedistributed among multiple interconnected processing systems that may belocal or remote to the processing system. As used herein, “diagnosticdevice” means a device capable of carrying out the L:M cone ratiomeasurements or L:M opsin gene haplotype determination to carry out themethods of invention, including but not limited to a microarray readeror a mass spectrometer.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light, of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1

Mutant OPN1LW and OPN1MW Genes in Bornholm Eye Disease

The first identified Yid-grade myopia locus was localized to chromosomeXq2.8 and designated MYP1 (M. Schwartz, M. Haim, D. Skarsholm, ClinicalGenetics 38, 281 (October, 1990)), The phenotype is also known as theBornholm Eye Disease (BED), and is an X-linked cone dysfunction syndromewith myopia, astigmatism and optic nerve changes (T. L. Young et al.Archives of Ophthalmology 122, 897 (June, 2004); U, Radhakrishna et al.,Investigative Ophthalmology & Visual Science supplement (abstract #3814)(2005); Michaelides et al., Ophthalmology 112, 1448 (2005)). Part of thephenotype of BED with X-linked cone dysfunction syndrome is an abnormalcone electroretinogram (ERG). The OPN1LW and OPN1MW genes reside at Xq28and encode cone photopigments responsible for the initial events thatgenerate the cone ERG.

The L and M Cone opsin genes were evaluated as candidates for the BEDphenotype. The two unrelated X-linked myopia/cone dysfunction familiesdescribed by Young et al. (T. L. Young et al., Archives of Ophthalmology122, 897 (June, 2004)) have color vision deficiencies which are causedby the absence of an OPN1MW gene in either of the first two positions inthe cone opsin gene array in the original BED (M. Schwartz, M. Haim, D.Skarsholm, Clinical Genetics 38, 281 (October, 1990)) family and by theabsence of an intact OPN1LW gene in the case of the Minnesota (MN)family. In a third family, residing in India, the affected males (U.Radhakrishna et al., Investigative Ophthalmology & Visual Sciencesupplement (abstract #3814) (2005)) have normal color vision. The firstgene in the X-chromosome opsin array was selectively amplified andindividual exons from affected and unaffected males in the MN, BED1, andIndian families were directly sequenced. The opsin genes downstream ofthe first gene were also selectively amplified, and the exons weredirectly sequenced. For all affected males in the MN family, the firstposition (5′-most) opsin gene in the array encoded an M opsin with anunusual combination of amino acids specified by the dimorphic codons inexon 3. This combination was Leucine at amino acid position 153 (L153),Valine at position 171 (V171), Alanine at 174 (A174), Valine at 178(V178), and Alanine at 180 (A180), henceforth abbreviated “LVAVA.” Thesecond gene in the array encoded a combination of amino acids at thesepositions (“MVVVA”) typically found in M opsins in individuals with novision abnormalities.

The affected members of the second, unrelated BED family (BED1) reportedby Young et al. (T. L. Young et al., Archives of Ophthalmology 122, 897(June, 2004)) and the Indian family (U. Radhakrishna et al.,Investigative Ophthalmology & Visual Science supplement (abstract #3814)(2005)) were also found to have the LVAVA combination, but in the Lopsin. In both of these latter families, the downstream genes inaffected males encoded variants that are typical of individuals withnormal vision. Unaffected males in the BED families did not have anLVAVA variant. As a control experiment, 261 OPN1MW genes and 320 OPN1LWfrom males with no serious vision abnormality were sequenced. None ofthe genes specified the LVAVA combination.

Affected males in five additional families (M. Michaelides et al.,Ophthalmology 112, 1448 (2005); M. McClements, M. Neitz, A. Moore, D. M.Hunt, Invest Ophthalmol Vis Sci, ARVO E (2010)) and one other unrelatedindividual with the BED phenotype were found to have either the LVAVAcombination or a similar combination, in which isoleucine is present atposition 171 (1171) instead of valine. This combination is designated“LIAVA” and was previously shown to cause photoreceptors to benon-functional in adults (J. Carroll, M. Neitz, H. Hofer, J. Neitz, D.R. Williams, Proceedings of the National Academy of Sciences of theUnited States of America 101, 8461 (2004), Neitz et al., VisualNeuroscience 21, 205 (2004); M. A. Crognale et al., Visual Neuroscience21, 197 (2004)). Affected members of a seventh family reported to haveX-linked cone dysfunction syndrome were found to have a mutation thatreplaces the cysteine normally found at position 203 with arginine(C203R) in both the L and M opsins (M. Michaelides et al., Ophthalmology112, 1448 (2005)), a mutation known to render the opsin non-functional(M. Michaelides et al., Ophthalmology 112, 1448 (2005); J. Winderickx etal., Nature Genetics 1, 251 (1992); Nathans et al., Science 245, 831(1989)).

Cone Phenotype of BED Opsin Mutation in Mice with a Targeted GeneReplacement

Although the LIAVA and C203R mutations found in some of the familieshave been previously documented to cause cone photoreceptor malfunction,the LVAVA amino acid combination found in many BED families and itsimpact on cone function and viability was never identified. Individualswith LVAVA encoded in their only expressed X-linked cone pigment genehave cone dystrophy indicating that cones expressing this haplotypefunction abnormally and eventually degenerate. To verify the abnormalcone function associated with LVAVA, a mouse line was created in whichexons 2 through 6 of the mouse M opsin gene were replaced with a cDNAcontaining exons 2-6 of a human L opsin gene that specified the LVAVAcombination. A control mouse line was also created that was identical inthe structure of the X-chromosome opsin gene replacement except that itspecified the combination LIAIS, which is commonly found in individualswith normal vision. The mice were tested using ON-OFF ERG using an Lcone isolating stimulus. The ERG amplitudes were reduced in mice withthe LVAVA mutation compared to control mice, consistent with theabnormal ERG findings in the BED patients (T. L. Young et al., Archivesof Ophthalmology 122, 897 (June, 2004)). The ERG-a-wave, the componentmost associated with photoreceptor function, was reduced in amplitude byhalf in the LVAVA mouse compared to the control mouse.

Cone Ratio and the Severity of the BED Phenotype

In the case of individuals with the LIAVA or C203R mutation, both ofwhich render cones expressing them non-functional, a single cone typeabsorbing in the middle-to-long wavelengths is left, accounting fortheir color vision defects. In the case of individuals with the LVAVAmutations and a color vision defect, cones containing the LVAVA opsinfunction, but the first two genes in array encode the same opsin type, Lfor the BED1 family, and M for the MN family. In contrast, in the Indianfamily, L cones express the abnormally functioning LVAVA photopigments,but a normal M opsin is expressed in a separate cone subpopulation andthe individuals with BED myopia in this family have normal color vision.

Usually, only the first two genes in the X-chromosome opsin gene arrayare expressed. However, the BED/X-linked high myopia patients have oneX-linked opsin gene with a mutation that causes cone photoreceptormalfunction and second normal gene. Each of the first two opsin genesfrom the array is expressed in its own submosaic of cones with the twobeing randomly interspersed. Each of the mutations found to beassociated with BED/X-linked high myopia produces a more debilitatingvision disorder (cone dystrophy in the cases of LVAVA) or one in which Land M cone function is absent entirely in adults (blue cone monochromacyin the case of LIAVA and C203R) when it is the only L/M opsin expressedin an individual's retina. What appears to rescue the high myopiapatients from the more debilitating retinal phenotype is the presence ofa normal X-chromosome pigment gene expressed in a submosaic of cones.However, having the interspersed normal and mutant cones appears to beresponsible for the high myopia.

There is widespread variability in L:M cone ratio in the normalpopulation. A similar variation in cone ratio was found among the LIAVABED subjects (FIG. 1). It is clear from the adaptive optics (AO) imagesthat the mutations associated with BED disrupt the cone mosaic, mostlikely impairing the ability of the eye to extract reliable informationabout the presence of sharply focused, fine-grained images fromcomparisons of activity among neighboring cones and thus interferes withemmetropization. Imaging of three individuals showed a dramaticillustration of how the degree of cone mosaic disruption correlated withaxial length and the severity of myopia (FIG. 1E).

In the LVAVA BED patients, the mutant cones are functional, but thedifference in response between normal and mutant cones is larger thanwould be produced by two normal cones, one on the light side and one onthe dark side of a sharply focused dark-light edge in an image. Inadulthood, cones containing an opsin with the LIAVA combination arecompletely non-functional (J. Carroll, M. Neitz, H. Hofer, J, Neitz, R.Williams, Proceedings of the National Academy of Sciences of the UnitedStates of America 101, 8461 (2004); M. Neitz et al., Visual Neuroscience21, 205 (2004; M. A. Crognale et al., Visual Neuroscience 21, 197(2004); however, there is evidence that they function to some degree inchildhood (L. Mizrahi-Meissonnier, S. Merin, E. Banin, D. Sharon,Investigative Ophthalmology and Visual Science (Mar. 20, 2010, 2010)).

Here, for the first time, the complete etiology for a form of myopia(i.e., Bornholm Eye Disease) was determined.

Example 2

Opsin Mutations and Haylotypes Associated with Myopia

Among humans with normal color vision there is tremendous variation inthe amino acid sequences of the L and M opsins that has arisen viaunequal homologous recombination (J. Nathans, T. P. Piantanida, R. L.Eddy. T. B. Shows. D. S. Hogness, Science 232, 203 (1986); M.Drummond-Born, S. S. Deeb A. G. Motulsky, Proceedings of the NationalAcademy of Sciences of the Untied States of America 86, 983 (1989); B.C. Verrelli, S. A. Tishkoff, American Journal of Human Genetics 75, 363(2004)). For example, in the control sample described above, there were34 different L opsin sequences in 320 subjects, and 17 different M opsinsequences in 261 subjects. The ratio of L to M cones also varies widelyamong humans. For example, among Caucasian males with normal colorvision, the ratio of L:M cones ranges hum 1.1:1 to 19:1, with an averageof 2.7:1 (J. Carroll, M. Neitz, J. Neitz, Journal of Vision 2, 531(2002); H. Hofer, J. Carroll, J. Neitz, M. Neitz, D. R. Williams,Journal of Neuroscience 25, 9669 (October, 2005)).

To determine if a biased L:M cone ratio would be protective againstmyopia, the mean axial length versus the mean L:M cone ratios for threeethnic groups were plotted. L:M cone ratios were estimated previouslyfrom ERGs and genetics for males self-reported to be Caucasian (n=86)(H. Hofer, J. Carroll, J. Neitz, M. Neitz, D. R. Williams, Journal ofNeuroscience 25, 9669 (October, 2005); J. Carroll, C. McMahon, M. Neitz,J. Neitz, Journal of the Optical Society of America A 17, 499 (March,2000)) and African (n=28) (C. McMahon, J. Carroll, S. Awua, J. Neitz, M.Neitz, Journal of Vision 8, 1 (2008)). The L:M ratio for a sample of 5unrelated Japanese males (n=5) was also determined. The values rangedfrom 48.13% L to 38% L cones, with an average of 43.4% L conescorresponding to a mean ratio of 0.8 L:1M. Even for this small samplethe results indicated a statistically significant difference (p<0.0001;Mann Whitney U) in the mean L:M cone ratio for Caucasian males versusJapanese males (FIG. 2). The mean axial length data were from Twelker etal. (J. D. Twelker et al., Optometry and Vision Science 86, 918 (2009))for boys age 12 at their last birthday in the ethnic categories White,African American, and Asian. The L:M cone ratio bias was stronglynegatively associated with axial length (R²=0.99), and thus withsusceptibility to myopia.

Variation in the coding sequences of the OPN1LW and OPN1MW genes wasthen evaluated as candidates for causing myopia. Subjects were 336self-reported Caucasian males, age 21 years or older, all of whom wereconfirmed to have normal color vision. Axial lengths and conicalcurvatures were measured using the Zeiss IOL master without cycloplegia,and their spherical equivalent refraction (SER) were calculated using anequation described in Methods below. An opsin gene haplotype wasdetermined for each subject by selectively amplifying and sequencingexons 2, 3 and 4 of the OPN1LW and OPN1MW genes. Haplotypes were createdusing the amino acids encoded by codons 65, 111, 116, 153, 171, 178,180, 230, 233, and 236 of the OPN1LW and OPN1MW genes. Completehaplotypes were obtained for 303 subjects. Haplotypes were identified asthe combination of amino acids at the variant positions encoded by exons2, 3 and 4. Over 50%, or 159 males, belonged to 13 haplotype groups withat least 3 subjects per group (see FIG. 3A). Within each of the 13haplotype groups there was no variation at codons 65, 111, 116, 230, or233 in either gene or in codon 236 in OPN1MW genes.

Within each haplotype, it was expected that subjects varied in coneratio, and subjects with a highly biased L:M cone ratio would beprotected from the myopia-genic action of the haplotype. The average SERfor each haplotype was calculated as the mean SER for the most myopichalf of the subjects within the haplotype. The most-myopic 50% from eachgroup were considered, based on the premise that these individuals wouldhave more nearly equal L:M cone ratios and be a more accurate reflectionof potential for each haplotype to cause myopia. The haplotypes werearranged in order of myopic potential with haplotype number 1 having theleast potential for causing myopia, and haplotype number 13 having thegreatest, and the myopic potential increased from an average SER of −1to −9 diopters (FIG. 3A). A one-way analysis of variance was used totest for an association between haplotype and spherical equivalentrefraction (SER); there was a highly significant association (p<0.0001).

The L:M cone ratio of eleven of the subjects from FIG. 3A was estimatedusing flicker photometric ERG and individualized cone spectra (J.Carroll, C. McMahon, M. Neitz, J. Neitz, Journal of the Optical Societyof America A 17, 499 (March, 2000)). For each of the 11 subjects, thepredicted SER was calculated by taking the mean SER for the haplotypegroup from FIG. 3A, and scaling it according to the percentage of L plusM cones that were L cones for each subject. For example, if a person hadhaplotype 8 (FIG. 3A), their myopic potential was −4.5 diopters. If thatperson had a 1:1 cone ratio they would be expected to have the full −4.5diopters of refractive error. However, if he had nearly 100 percent Lcones he would be expected to be nearly emmetropic. 75% L cones fallsmidway between a 1:1 cone ratio (50% L) and 100% L so a person withhaplotype 8 and 75% L cones would be predicted to have 50% of the SER(or −4.5/2=2.25 diopters).

The SER for each subject was compared to the SER predicted by thecombined haplotype and cone ratio data (FIG. 3B). The correlationcoefficient (R²) was 0.86, suggesting that 86% of the SER could bepredicted by knowing the Xq28 opsin gene locus haplotype and the L:Mcone ratios for each subject in this sample. L:M cone ratio is alsoencoded by genetic variation in the X-linked opsin gene array.

Example 3

Red Content of Video Games Causes Increased Refractive Error

The potential for the red content of video games to contribute to myopiawas evaluated as follows. Baseline axial length measurements wereobtained for seven 18 year old subjects, and at 2 week intervalsthereafter, for 2 months during which time each subject played a videogame for 1 hour per day while wearing special goggles. The video gamewas in black and white, and while playing the game, subjects viewed thecomputer monitor through a pair of goggles in which the right lens wasclear so that the L and M cones were nearly equally activated, and theleft lens was tinted such that only the L but not the M cones wereactivated. The data plotted in FIG. 4A shows the trend line of asignificant myopic shift (p=0.0076) in the left eye that viewed the redvideo games relative to the right control eye of the subjects. Theincreased axial length of eye exposed to the red relative to black andwhite video game corresponds to an increase in refractive error of ⅓ ofa diopter per year.

Example 4

Glasses that Control the Spectral Distribution of Light Reaching theRetina can Prevent Myopia

The ability of modified eyeglasses to influence the growth of the axiallength of the eye when routinely worn by children was evaluated. Bothlenses of the study eyeglasses had the optimal correction for eachsubject as determined by the participant's optometrist. One lens in eachpair of glasses was the experimental lens, which was tinted and had aholographic diffuser applied to the surface. The tint removed red lightand the diffuser spread the incident light rays over an angle of 0.5degree to produce a slight blur to reduce activity differences betweenadjacent cones. The other lens in each pair of glasses was a controllens that was tinted with a neutral filter that equally activated L andM cones, and was chosen so that both eyes were exposed to the same lightintensity. The dominant eye was identified for each subject, and for thefirst 3 month period, all subjects wore the experimental lens on thedominant eye. Subjects were offered the opportunity to re-enroll after 3months, and those who chose to re-enroll wore the experimental lens overthe non-dominant eye during the second 3 month period.

Before participants began wearing the experimental glasses, the axiallengths of both eyes were measured using the Zeiss IOL Master, which hasbeen established previously to produce accurate and reproduciblemeasurements in children, with a standard deviation between repeatedmeasures of axial length in children of ±0.019 (A. Carkeet, S. M. Saw,G. Gazzard, W. Tang, D. T. Tan, Optometry and Vision Science 81, 829(2004); J. Gwiazda et al., Investigative Ophthalmology & Visual Science44, 1492 (2003)). Each axial length measurement plotted in FIG. 4 wasthe average of twenty measurements for each eye. General baselinecharacteristics of the thirteen subjects enrolled in the study are givenin Table 2. Spherical equivalent refraction (SER) was determined forboth eyes at the beginning of the study, and axial length measurementswere determined for each eye the day that the children received theirmodified eye glasses. The values given were the average of twentymeasurements for each eye. The last column indicates which eye had theexperimental Versus control lens (OS left eye, OD right eye).

TABLE 2 Axial Axial Subject Length Length Experi- ID SER SER (mm) (mm)mental No. Gener Age (OD) (OS) (OD) (OS) Control 001 F 12 −1.50 −1.5025.18 25.27 OD/OS 002 F 13 −3.75 −3.25 24.76 24.95 OS/OD 003 F 14 −7.875−8.125 27.53 27.84 OD/OS 004 M 11 −3.25 −3.25 23.96 23.93 OS/OD 005 F 9−2.75 −3.00 24.80 24.86 OS/OD OD/OS 006 F 11 −1.50 −1.375 22.93 23.08OS/OD OD/OS 007 M 8 −1.50 −1.50 25.25 25.17 OD/OS OS/OD 008 M 13 −1.75−1.50 24.39 24.54 OD/OS OS/OD 009 F 10 −2.125 −2.125 25.88 25.98 OS/ODOD/OS 010 F 11 −1.375 −1.375 24.15 24.10 OS/OD 011 M 8 −1.50 −1.50 24.0824.09 OD/OS 012 M 11 −1.125 −1.25 23.21 23.46 OS/OD OD/OS 013 M 12 −4.00−3.75 25.85 25.77 OS/OD OD/OS

All participants completed the study with the dominant eye as theexperimental eye and the other eye as an internal control. Sevenparticipants re-enrolled to complete the study a second time, but withthe experimental lens on the non-dominant eye. Which lens, andtherefore, which eye, had the experimental versus control lens is listedin Table 2. Initial, spherical equivalent refraction (SER) was measuredby cycloplegic autorefraction to determine eligibility for the study,and it ranged from a minimum of −1.00 to a maximum of −8.50 diopters.Baseline axial lengths ranged from 22.93 to 27.53 millimeters (mm) forthe right eye (OD) and from 23.08 to 27.84 mm for the left eye (OS).

Axial length growth was the primary outcome measure used to evaluate theeffect of the experimental lens versus the control lens over the courseof three months. The relative growth of axial length was determined forthe twenty eyes wearing the experimental lens and for the twenty eyeswearing the control lens. Growth curves for each of the twenty trialsdemonstrated the dramatic difference in the experimental versus thecontrol group (FIGS. 4B and C). Growth curves for eyes that wore theexperimental lens clustered around baseline representing a reduction inelongation of the eye, whereas growth curves for eyes that wore thecontrol lens deviated toward positive growth, representing continuedelongation of the eye. Sixteen of the twenty trials followed this growthpattern, where the experimental lens reduced growth and the control lenshad continued growth. Overall, normalized differences in axial lengthbetween the control and experimental eyes were evaluated by a paired2-sample t test. Absolute difference in growth between the two eyesreached statistical significance by day 30, as a group. Individually,the date where the growth difference between the two eyes reachedsignificance ranged from day 30 to day 75.

The rate of axial elongation tier eyes wearing experimental versuscontrol lenses was also evaluated (FIG. 4D). The average axial lengthgrowth rate in the eyes wearing the experimental lens was 0.063±0.33μm/day (mean±SE), whereas the average axial length growth rate in theeyes wearing the control lens was 1.43±0.24 μm/day. Again, sixteen ofthe twenty trials resulted in reduced rate of axial elongation for theeye wearing the experimental lens versus the eye wearing the controllens. Reduction in the overall growth rate in the experimental grouprelative to the control group was statistically significant (p=0.01019,FIG. 4D).

On average, the eyes wearing the experimental lens grew nearly ten timesslower than eyes wearing the control lens.

METHODS

Color Vision Testing: Participants were screened for the presence of aninherited red-green color vision deficiency using the Nagel anomaloscopeand the Richmond HRR 2004 edition pseudoisochromatic plate test.

Determination of L:M cone ratios cone ratios were estimated usingflicker photometry and genetics as previously described (H. Hofer, J.Carroll, J. Neitz, M. Neitz, D. R. Williams, Journal of Neuroscience 25,9669 (October, 2005); J. Carroll, C. McMahon, M. Neitz, J. Neitz,Journal of the Optical Society of America A 17, 499 (March, 2000)).

Adaptive Optics Imaging: Images of retinas were obtained using adaptiveoptics as described previously (J. Carroll, M. Neitz, H. Hofer, J.Neitz, D. R. Williams, Proceedings of the National Academy of Sciencesof the United States of America 101, 8461 (2004); J. Carroll et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 106, 20948 (2009); J. Carroll et al., Proc. Natl. Acad. Sci. USAsubmitted (2010)).

Axial length, corneal curvature, and spherical equivalent refraction(SER): Axial lengths and corneal curvatures for both eyes will bemeasured for each subject using the Zeiss IOL Master, and the predictedspherical equivalent refraction (SERs) were calculated using a formuladerived from a linear regression of a dataset of actual SERs, axiallengths (AL) and corneal curvatures (CC) from a group of 400 malesubjects. The formula is: SER=−(AL*2.03+0.94*CC)+88.58, where the valuefor AL was the average of 20 measurements per eye, and CC was theaverage of two different methods of measuring corneal curvature.Measurements were made for both eyes.

Genetic analysis: DNA was isolated from whole blood or from buccal swabsuse the PureGene kit. The polymerase chain reaction was used toselectively amplify the OPN1LW and the OPN1MW genes, and exons 2, 3, and4 were directly sequenced as previously described (M. Neitz et al.,Visual Neuroscience 21, 205 (2000)). Quantitative real time PCR wasperformed on a DNA sample from each subject to estimate the relativenumber of OPN1LW and OPN1MW genes using previously described assays (M.Neitz, J. Neitz, Color Research & Application 26, S239 (2001)).

Human Subjects Research: All human subjects research was conducted underIRB approved protocols at the Medical College of Wisconsin and followedthe tenets of the Declaration of Helsinki.

Knock-in/Knock-out mouse constructs. The targeting vector was designedto replace the endogenous mouse OPN1MW gene on the X-chromosome with ahuman L opsin cDNA. The 5′ homology arm was 11,917 bp in length extendsfrom nucleotide position 71,366,218 which is upstream of the OPN1MW geneon the mouse X-chromosome through codon 65 of exon 2 of the mouse OPN1MWgene (nucleotide position 71,378,135 July 2007 version of mouse genomeassembly). Site directed mutagenesis (QuickChange Kit, Stratagene) wasused to alter mouse codons 58, 62, and 65 to encode the same amino acidsas the corresponding codons in human OPN1LW. Amino acids 58 and 62 donot vary among human OPN1LWs but codons 65 does, and in our constructthis codon specifies threonine (T65). Mouse codon 58 was changed fromACC to GTC, mouse codon 62 was changed from CIT to TTT, and mouse codon65 was changed from GTT to ACT. A human cDNA segment from plasmid hs7(M. Drummond-Borg, S. S. Deeb, A. G. Motulsky, Proceedings of theNational Academy or Sciences of the United States of America 86, 981(1989)) extending from codon 66 through the polyadenylation signal(nucleotide 1679 in plasmid hs7 plus 142 base pairs of the polylinkerfrom hs7 was ligated in frame to the 5′ homology arm. A PGK-NEO cassetteflanked by lox P sites was ligated downstream of the human cDNAfragment, and downstream of that was ligated the 3′ homology armextending from mouse X-chromosome nucleotide 71,389,460 to 71,392,250.The 3′ homology arm corresponds to a 2823 base pair segment withinintron 5 of the mouse OPN1MW gene. All vectors were confirmed by directsequencing of the complete vector. Creation of the final vector used andof the knock-in/knock-out mice was done by Ozgene Inc. The targetingconstructs were electroporated into embryonic stem cells, and Neomycinresistant cells were screened by Southern Hybridization for correctlytargeted events and confirmed by sequencing. Mice showing germlinetransmission of the correctly targeted locus were mated to Cre mice todelete the PGK Neo cassette. Animals were screened by PCR for the finalaltered locus, and confirmed by direct sequencing. Upon receivingfounder mice from Ozgene, genomic DNA from each mouse was sequenced toconfirm the presence of the correctly targeted locus.

Gene expression at the targeted locus was controlled by the endogenousmouse regulatory DNA sequences, and the N terminal tail of the encodedopsin corresponded to that encoded by mouse exon 1. The portion of the Nterminus encoded by exon 1 differed from human in that amino acids 4thru 8 were deleted and the sequence differed at 7 other positions asfollows: threonine instead of alanine at position 11, glutamic acidinstead of arginine at position 13, glutamine instead of histidine atposition 14, threonine in place of proline at position 15, leucineinstead of glutamine at position 16, histidine instead of serine atposition 18, and lysine instead of arginine at position 37. Human Lopsins vary at amino acid positions 65, 111 and 116 encoded by exon 2and 230, 233 and 236 encoded by exon 4. The targeted locus specifiedT65, I116, S116, I230, A233 and M236. Two versions of the targeted locuswere constructed regarding the amino acid sequence specified by exon 3.The control locus specified L153, I171, A174, I178, S180 (LIAIS) whichcorresponds to the sequence found in chimpanzee L opsins, and mutantunder study specified L153, V171, A174, V178, A180 (LVAVA).

Mouse ERGs: Mice were anesthetized with ketamine/xylazine and kept on awarming table throughout the experiment. The recording electrode wasplaced on the cornea, the reference electrode was placed under the lidand the ground electrode was touching the tongue. ON-OFF ERGs(alternating 30s ON 30s OFF) were performed using 525 nm LED stimuli at5 different light intensities (0.3 log intensity steps) controlled bypulse width modulation. The 525 nm lights produce responses mediated byhuman L cone opsin encoded by the transgenes but not endogenous mouse UVopsin. Recording was performed under light adapted conditions in whichrods were saturated.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

What is claimed is:
 1. A method of determining the opsin haplotype of asubject, said method comprising: (a) detecting opsin haplotype 1 as setforth in Table 1 in a biological sample obtained from the subject,wherein said haplotype 1 comprises an L-opsin gene encoding an L-opsinpolypeptide having a methionine (M) at residue 153, an isoleucine (I) atresidue 171, an I at residue 178, a serine (S) at residue 180, and an Mat residue 236, and an M-opsin gene encoding an M-opsin polypeptidehaving an M at residue 153, a valine (V) at residue 171, a V at residue178, and an alanine (A) at residue 180; and wherein said detectingcomprises (i) amplifying and sequencing exons 2, 3, and 4 of the L-opsinopsin gene, and (ii) amplifying and sequencing exons 2, 3, and 4 of theM-opsin opsin gene.
 2. The method of claim 1, wherein said biologicalsample is selected from the group consisting of blood, saliva, cellsfrom buccal swabbing, biopsies of skin, and amniotic fluid.
 3. Themethod of claim 1, further comprising purifying nucleic acids from saidbiological sample.
 4. The method of claim 3, wherein said nucleic acidscomprise genomic DNA.
 5. The method of claim 1, wherein said amplifyingsteps comprise polymerase chain reaction (PCR).
 6. The method of claim1, further comprising: determining the ratio of L-cones to M-cones inthe subject, wherein said determining comprises an electroretinogram. 7.The method of claim 6, wherein said electroretinogram is a flickerphotometric electroretinogram.
 8. A method comprising: (a) detectinghaplotype 1 as set forth in Table 1 in a biological sample obtained froma subject, wherein said haplotype 1 comprises an L-opsin gene encodingan L-opsin polypeptide having a M at residue 153, an I at residue 171,an I at residue 178, a S at residue 180, and an M at residue 236, and anM-opsin gene encoding an M-opsin polypeptide having an M at residue 153,a Vat residue 171, a Vat residue 178, and an A at residue 180; (b)determining the subject is susceptible to increased myopic potentialwhen haplotype 1 is detected; and (c) treating the subject determined tohave increased myopic potential with at least one lens that reducesmyopia progression.
 9. The method of claim 8, wherein said lens is aglasses lens or a contact lens.
 10. The method of claim 8, wherein saidlens is a blur-inducing lens.
 11. The method of claim 8, wherein saidlens comprises a holographic diffuser.
 12. The method of claim 11,wherein said holographic diffuser is applied to a surface of said lens.13. The method of claim 8, wherein said lens is tinted.
 14. The methodof claim 1, said method further comprising detecting one of haplotypes 2to 13 as set forth in Table 1 in the biological sample obtained from thesubject, wherein said haplotype 2 comprises an L-opsin gene encoding anL-opsin polypeptide having a M at position 153, a V at position 171, anI at position 178, a S at position 180, and a M at position 236, and anM-opsin gene encoding an M-opsin polypeptide having a M at position 153,a V at position 171 and at position 178, and an A at position 180;wherein said haplotype 3 comprises an L-opsin gene encoding an L-opsinpolypeptide having a leucine (L) at position 153, V at position 171, anI at position 178, a S at position 180, and a M at position 236, and anM-opsin gene encoding an M-opsin polypeptide having a M at position 153,a V at position 171 and at position 178, and an A at position 180;wherein said haplotype 4 comprises an L-opsin gene encoding an L-opsinpolypeptide having a M at position 153, a V at position 171, an I atposition 178, a S at position 180, and a M at position 236, and anM-opsin gene encoding an M-opsin polypeptide having a M at position 153,a V at position 171, an I at position 178, and an A at position 180;wherein said haplotype 5 comprises an L-opsin gene encoding an L-opsinpolypeptide having a L at position 153, a V at position 171, an I atposition 178, an A at position 180, and a M at position 236, and anM-opsin gene encoding an M-opsin polypeptide having a M at position 153,a V at position 171, an I at position 178, and an A at position 180;wherein said haplotype 6 comprises an L-opsin gene encoding an L-opsinpolypeptide having a M at position 153, a V at position 171, an I atposition 178, an A at position 180, and a M at position 236, and anM-opsin gene encoding an M-opsin polypeptide having a M at position 153,a V at position 171, an I at position 178, and an A at position 180;wherein said haplotype 7 comprises an L-opsin gene encoding an L-opsinpolypeptide having a L at position 153, a V at position 171, an I atposition 178, a S at position 180, and a M at position 236, and anM-opsin gene encoding an M-opsin polypeptide having a L or a M atposition 153, a V at position 171, an I at position 178, and an A atposition 180; wherein said haplotype 8 comprises an L-opsin geneencoding an L-opsin polypeptide having a L at position 153, a V atposition 171, an I at position 178, a S at position 180, and a M atposition 236, and an M-opsin gene encoding an M-opsin polypeptide havinga M at position 153, a V at position 171, an I at position 178, and an Aat position 180; wherein said haplotype 9 comprises an L-opsin geneencoding an L-opsin polypeptide having a L at position 153, an I atposition 171, an I at position 178, a S at position 180, and a M atposition 236, and an M-opsin gene encoding an M-opsin polypeptide havinga M at position 153, a V at position 171, a V at position 178, and an Aat position 180; wherein said haplotype 10 comprises an L-opsin geneencoding an L-opsin polypeptide having a M at position 153, a V atposition 171, a V at position 178, an A at position 180, and a V atposition 236, and an M-opsin gene encoding an M-opsin polypeptide havinga M at position 153, a V at position 171, an I at position 178, and an Aat position 180; wherein said haplotype 11 comprises an L-opsin geneencoding an L-opsin polypeptide having a M at position 153, a V atposition 171, an I at position 178, a S at position 180, and a V atposition 236, and an M-opsin gene encoding an M-opsin polypeptide havinga M at position 153, a V at position 171, a V at position 178, and an Aat position 180; wherein said haplotype 12 comprises an L-opsin geneencoding an L-opsin polypeptide having a L at position 153, a V atposition 171, an I at position 178, a S at position 180, and a M atposition 236, and an M-opsin gene encoding an M-opsin polypeptide havinga L at position 153, a V at position 171, an I at position 178, and a Sat position 180; wherein said haplotype 13 comprises an L-opsin geneencoding an L-opsin polypeptide having a L at position 153, a V atposition 171, an I at position 178, an A at position 180, and a M atposition 236, and an M-opsin gene encoding an M-opsin polypeptide havinga L or a M at position 153, a V at position 171, an I at position 178,and an A at position 180; and wherein said detecting comprises (i)amplifying and sequencing exons 2, 3, and 4 of the OPN1LW gene in anopsin array, and (ii) amplifying and sequencing exons 2, 3, and 4 of theOPN1MW gene in an opsin array.
 15. The method of claim 8, said methodfurther comprising: detecting one of haplotypes 2 to 13 as set forth inTable 1 in the biological sample obtained from the subject; wherein saidhaplotype 2 comprises an L-opsin gene encoding an L-opsin polypeptidehaving a M at position 153, a V at position 171, an I at position 178, aS at position 180, and a M at position 236, and an M-opsin gene encodingan M-opsin polypeptide having a M at position 153, a V at position 171and at position 178, and an A at position 180; wherein said haplotype 3comprises an L-opsin gene encoding an L-opsin polypeptide having aleucine (L) at position 153, V at position 171, an I at position 178, aS at position 180, and a M at position 236, and an M-opsin gene encodingan M-opsin polypeptide having a M at position 153, a V at position 171and at position 178, and an A at position 180; wherein said haplotype 4comprises an L-opsin gene encoding an L-opsin polypeptide having a M atposition 153, a V at position 171, an I at position 178, a S at position180, and a M at position 236, and an M-opsin gene encoding an M-opsinpolypeptide having a M at position 153, a V at position 171, an I atposition 178, and an A at position 180; wherein said haplotype 5comprises an L-opsin gene encoding an L-opsin polypeptide having a L atposition 153, a V at position 171, an I at position 178, an A atposition 180, and a M at position 236, and an M-opsin gene encoding anM-opsin polypeptide having a M at position 153, a V at position 171, anI at position 178, and an A at position 180; wherein said haplotype 6comprises an L-opsin gene encoding an L-opsin polypeptide having a M atposition 153, a V at position 171, an I at position 178, an A atposition 180, and a M at position 236, and an M-opsin gene encoding anM-opsin polypeptide having a M at position 153, a V at position 171, anI at position 178, and an A at position 180; wherein said haplotype 7comprises an L-opsin gene encoding an L-opsin polypeptide having a L atposition 153, a V at position 171, an I at position 178, a S at position180, and a M at position 236, and an M-opsin gene encoding an M-opsinpolypeptide having a L or a M at position 153, a V at position 171, an Iat position 178, and an A at position 180; wherein said haplotype 8comprises an L-opsin gene encoding an L-opsin polypeptide having a L atposition 153, a V at position 171, an I at position 178, a S at position180, and a M at position 236, and an M-opsin gene encoding an M-opsinpolypeptide having a M at position 153, a V at position 171, an I atposition 178, and an A at position 180; wherein said haplotype 9comprises an L-opsin gene encoding an L-opsin polypeptide having a L atposition 153, an I at position 171, an I at position 178, a S atposition 180, and a M at position 236, and an M-opsin gene encoding anM-opsin polypeptide having a M at position 153, a V at position 171, a Vat position 178, and an A at position 180; wherein said haplotype 10comprises an L-opsin gene encoding an L-opsin polypeptide having a M atposition 153, a V at position 171, a V at position 178, an A at position180, and a V at position 236, and an M-opsin gene encoding an M-opsinpolypeptide having a M at position 153, a V at position 171, an I atposition 178, and an A at position 180; wherein said haplotype 11comprises an L-opsin gene encoding an L-opsin polypeptide having a M atposition 153, a V at position 171, an I at position 178, a S at position180, and a V at position 236, and an M-opsin gene encoding an M-opsinpolypeptide having a M at position 153, a V at position 171, a V atposition 178, and an A at position 180; wherein said haplotype 12comprises an L-opsin gene encoding an L-opsin polypeptide having a L atposition 153, a V at position 171, an I at position 178, a S at position180, and a M at position 236, and an M-opsin gene encoding an M-opsinpolypeptide having a L at position 153, a V at position 171, an I atposition 178, and a S at position 180; wherein said haplotype 13comprises an L-opsin gene encoding an L-opsin polypeptide having a L atposition 153, a V at position 171, an I at position 178, an A atposition 180, and a M at position 236, and an M-opsin gene encoding anM-opsin polypeptide having a L or a M at position 153, a V at position171, an I at position 178, and an A at position 180; and determining thesubject is susceptible to increased myopic potential when one ofhaplotypes 2 to 13 as set forth in Table 1 is detected.